Enzymatic Methods and Enzymes

ABSTRACT

Provided are methods to identify modulators and in particular inhibitors of body malodour formation employing peptidase enzymes, the peptidase enzymes and corresponding nucleotide sequences, expression vectors, transfected host cells, methods of forming the peptidase enzymes and methods to prevent body malodour.

TECHNICAL FIELD

Provided are methods to identify modulators of body malodour formationemploying nucleotides encoding peptidases, constructs for peptidaseexpression and cells containing such constructs, and the resultingpeptidases.

The nucleotides code for proteins which are peptidase enzymes. Thepeptidases are involved in malodour formation and can be employed inmethods to identify modulators, in particular inhibitors, of bodymalodour formation. These inhibitors have the ability to prevent orreduce malodour formation on the body, in particular the human body.

BACKGROUND

Since the 1950s it is known that sweat secreted by the apocrine glandsis odourless and the undesirable smell of sweat only develops throughbacterial action. Accordingly, it was concluded that sweat containsmalodour precursors and malodourants are released through the enzymaticaction of bacteria on said precursors.

Body malodour, including in particular axilla malodour, is due to threemain classes of compounds: Sulfanylalcohols, unsaturated or hydroxylatedacids, and steroids. The methods described herein are useful to identifyinhibitors that prevent the formation of sulfanylalcohols.

Applicant previously identified the malodour precursor for theunsaturated or hydroxylated acids, and the aminoacylase enzyme (“AMRE”)forming them (EP 1258531). The AMRE enzyme can be used to screen forinhibitors of the malodour forming enzymes and thereby to identifycompounds to prevent or reduce body malodour.

For the release of sulfanylalcohols, applicant identified acystathionine-β-lyase from Corynebacterium sp. Ax20 and a precursorcompound (A. Natsch et al., Chemistry & Biodiversity 2004, 1058).

Starkenmann et al. postulated a different β-lyase from Staphylococcushaemolyticus releasing sulfanylalcohols from a different precursor,namely the precursor of formula FIII (WO2006079934).

While Staphylococcus bacteriae are generally not believed to be involvedin malodour formation to a major extent, the compound of FIII (in theexamples: “cys-gly-conjugate”) seems to contribute to body malodournonetheless.

The sequence SEQ ID NO: 3, and SEQ ID NO:4 as its hypothetical geneproduct, were previously published in a sequence database, but theprotein function, catalytic activity and its involvement in malodourgeneration were not known.

Applicant has now surprisingly identified a third type of enzyme(peptidases including but not limited to SEQ ID NO:2 and SEQ ID NO:4)that releases a precursor II from a precursor I (for example, withoutlimitation, compound of FIII) or alternatively, various other substratesdescribed herein below, and together with the action of the previouslyidentified cystationin-β-lyase finally forms a sulfanyl alcoholmalodourant (compare FIG. 1).

Both the novel type of peptidase and the β-lyase previously described byapplicants occur in Corynebacteria. Without wishing to be bound bytheory, it is generally accepted that highly unpleasant malodour isreleased from fresh sweat mainly by Corynebacteria.

Applicant showed that no single fraction of C. sp. Ax20 extract was ableto release malodourant from the precursor I, in particular, withoutlimitation, compound FIII. This demonstrates that there is no singleenzyme present in C. sp. extracts that can cleave precursor I (compareexamples herein-below, in particular example 11). Further applicantshowed that two enzymes are mediating the cleavage of precursor I, inparticular, without limitation, compound of FIII: first a peptidase (forexample, without limitation, SEQ ID NO: 2 or NO:4) cleaving thedipeptide between the gly and the cys residue, and then a β-Lyasesubsequently releasing the malodourant from the cys-conjugate (FIV)formed by the peptidase (compare examples herein-below, in particularexample 12).

The identified type of enzyme is useful as an alternative screeningtarget to identify inhibitors of malodour formation by sulfanyl alcoholmalodourants.

Precursor I (physiologically relevant substrates) has the generalformula FII

wherein R1 is selected from a group of alkane residues consisting ofmethyl, ethyl, propyl, butyl, pentyl, and hexyland wherein R2 and R3 are independently selected from the groupconsisting of H and methyl.

Accordingly, the sulfanylalkanol malodourant has the general formula FV

wherein R1 is selected from a group of alkane residues consisting ofmethyl, ethyl, propyl, butyl, pentyl, and hexyland wherein R2 and R3 are independently selected from the groupconsisting of H and methyl.

The peptidases will also react non-physiological substrates of formulaFI as detailed herein-below under the section “substrates of peptidase”,that are useful for screening purposes.

The type of peptidase enzyme is particularly interesting since, withoutwishing to be bound by theory, not only does it occur in the mostrelevant genus of bacteria, but this type of peptidase enzymes seem toperform the rate limiting step in the malodour forming enzymaticreactions, and the identified inhibitors can therefore expected to beparticularly effective.

Still further, again without wishing to be bound by theory, bothembodiments of peptidase (SEQ ID NO: 2 and SEQ ID NO: 4) belong to thesame class of enzymes (metallopeptidases) as the previously identifiedAMRE enzyme that forms malodourous unsaturated or hydroxylated acids,and screening with the peptidase enzymes (including, without limitation,enzymes of SEQ ID 2 and SEQ ID 4) may result in finding inhibitors alsoactive against the metallopeptidases involved in the formation ofmalodourous unsaturated or hydroxylated acids and therefore effective toreduce formation of malodourants of both classes at the same time.

SUMMARY

Provided is the following:

(1) A method to identify a modulator of body malodour formation, themethod comprising the steps of:

(i) contacting a peptidase with a peptidase substrate and at least onetest agent; and(ii) determining the effect of the at least one test agent on thepeptidase-mediated reaction rate,wherein the peptidase has the catalytic activity to release glycine fromthe substrate compound of formula FIII

wherein the peptidase is homologous to a polypeptide sequence selectedfrom the group consisting of SEQ ID NO:2 and SEQ ID NO:4 with a sequenceidentity of at least 40%;wherein the peptidase comprises the following conserved partialsequences:ERDGRWYGRGXADCKG between aminoacid 105 and 150,EGSEEXG between aminoacid 150 and 180,HSGXXGGXAPDA between aminoacid 205 and 255,GGSIPL between aminoacid 385 and 425;and wherein the aminoacids are numbered starting from the N-terminus ofthe substantially homologous peptidase in its naturally occurring form,and the letters refer to the single character aminoacid code and X isany one of the 20 common aminoacids.

(2) The method of item (1) wherein the peptidase substrate in step (i)is a compound of formula FI

wherein X is selected from the group consisting of S and O, and whereinR1 is residue selected from H and methyl, and wherein R2 is a residueselected from the group consisting of a straight or branched C1 to C10alkyl, a straight or branched C1 to C10 alkanol, a phenyl, and a benzyl.

In an alternative embodiment of item (2), X is S.

(3) The method of item (1) wherein the substrate is a compound selectedfrom the group consisting of S-benzyl-Cys-Gly, O-benzyl-Ser-Gly,(1-(2-hydroxyethyl)-1-methylbutyl)-L-cysteinyl-glycine,S-benzyl-Cys-Ala, O-benzyl-Ser-Ala, Pro-Gly, Ala-Gly, Ala-Ala, andPro-Ala

In an alternative embodiment of item (3), the substrate is a compoundselected from the group consisting of S-benzyl-Cys-Gly,O-benzyl-Ser-Gly,(1-(2-hydroxyethyl)-1-methylbutyl)-L-cysteinyl-glycine,S-benzyl-Cys-Ala, and O-benzyl-Ser-Ala.

In an alternative embodiment of item (3), the substrate is a compoundselected from the group consisting of Pro-Gly, Ala-Gly, Ala-Ala,Pro-Ala.

(4) The method of any one of items (1) to (3) wherein an additionalenzyme, a cystathionine-β-lyase, is incubated with the substrate and thetest agent in parallel or subsequently, and wherein in step (ii), theeffect of the test agent on cleavage by the peptidase and β-lyaseenzymes is determined by the change in formation of at least one oftheir reaction products, optionally a reaction product selected from athiol reaction product and a hydroxy reaction product.

(5) A kit comprising:

(i) a peptidase as described herein, in particular as defined under item(1), and(ii) a substrate compound that is cleaved by the peptidase, for combineduse to identify test agents as modulators of the peptidase and malodourformation.

In one particular embodiment of item (5), the substrate compound is acompound of formula FI

wherein X is selected from the group consisting of S and O, and whereinR1 is residue selected from H and methyl, and wherein R2 is a residueselected from the group consisting of a straight or branched C1 to C10alkyl, a straight or branched C1 to C10 alkanol, a phenyl, and a benzyl.

In another embodiment of item (5), said substrate compound of formula FIis selected from the group consisting of S-benzyl-Cys-Gly,O-benzyl-Ser-Gly,(1-(2-hydroxyethyl)-1-methylbutyl)-L-cysteinyl-glycine,S-benzyl-Cys-Ala, O-benzyl-Ser-Ala, Pro-Gly, Ala-Gly, Ala-Ala, andPro-Ala.

In yet another embodiment of item (5), said substrate compound offormula FI is selected from the group consisting of S-benzyl-Cys-Gly,O-benzyl-Ser-Gly,(1-(2-hydroxyethyl)-1-methylbutyl)-L-cysteinyl-glycine,S-benzyl-Cys-Ala, O-benzyl-Ser-Ala.

In yet another embodiment of item (5), said substrate compound offormula FI is selected from the group consisting of Pro-Gly, Ala-Gly,Ala-Ala, and Pro-Ala.

(6) A method of inhibiting the peptidase as defined herein, inparticular under item (1), in its ability to cleave its substratewherein the peptidase is contacted with a peptidase inhibitor.

(7) The method of item (6) for preventing or reducing the formation ofbody malodour wherein an inhibitor of a peptidase is applied to a bodysurface, and wherein the peptidase is a peptidase as described herein,in particular, without limitation, under item (1).

(8) The method of item (7) wherein the compound is applied in form of adermatologically acceptable composition comprising at least oneexcipient.

(9) A method for preparing a personal care product having an effectagainst body malodour formation wherein an inhibitor of a peptidase isadded to a personal care product formulation, and wherein the peptidaseis a peptidase as described herein, in particular, without limitation,under item (1).

(10) A composition comprising a peptidase substrate and an isolatedpeptidase,

wherein the peptidase is the peptidase as described herein, inparticular, without limitation, under item (1);wherein the peptidase substrate is the peptidase substrate as describedherein, in particular under any one of items (2) or (3) or any one oftheir particular embodiments.

(11) The composition of item (10) wherein the peptidase is in a formselected from the group consisting of in isolated form, in form of apreparation containing functional peptidase, in form of heterologousexpression in a suitable host cell, in form of Corynebacterium jeikeiumexpressing the peptidase, and in form of Corynebacterium jeikeium K411expressing the peptidase.

(12) An isolated peptidase wherein the peptidase is the peptidase asherein described, in particular, without limitation, as described underitem (1),

and wherein the peptidase is homologous to a sequence selected from SEQID NO:2 and SEQ ID NO: 4 with a sequence identity of at least 40%.

(13) The isolated peptidase of item (12) wherein the peptidase ishomologous to a sequence selected from SEQ ID NO:2 and SEQ ID NO: 4 witha sequence identity of at least 80%.

In other embodiments, the sequence identity is at least 85%, at least90%, at least 95%, or at least 98%.

(14) A nucleotide encoding a peptidase wherein the peptidase is as thepeptidase as described herein, in particular, without limitation, underitem (1), which is selected from the group consisting of

a nucleotide substantially homologous to a nucleotide sequence of SEQ IDNO:1 as determined by sequence identity,

a nucleotide which is a conservatively modified variant of SEQ ID NO:1not causing aminoacid changes when translated into the correspondingprotein,

a nucleotide substantially homologous to SEQ ID NO:1 as determined byhybridisation,

wherein the substantially homologous nucleotide as determined bysequence identity has a sequence identity of at least 80%.

In other embodiments, the sequence identity is at least 85%, at least90%, at least 95%, or at least 98%.

(15) The isolated nucleotide of item (16) wherein said isolatednucleotide forms part of an expression vector.

(16) The isolated nucleotide of item (16) wherein the expression vectorforms part of a host cell transfected with the expression vector.

(17) A method of forming a peptidase comprising the step of culturinghost cells comprising an expression vector encoding for the peptidaseunder conditions sufficient for expression, thereby forming thepeptidase and optionally recovering it from the cells, wherein thepeptidase is the peptidase as herein described, in particular, withoutlimitation, as described under item (1).

DETAILED DESCRIPTION

The following passages describe in detail the peptidase and itsvariants, its substrates, its reaction products, its use in theidentification of modulators and inhibitors of body malodour includingvarious screening assays and how to perform them, including the cellsthat may be used, assays using purified peptidase, peptidasetranscription assays, expression systems for peptidase, overexpressionof peptidase, transfection of peptidase constructs into cells, peptidaseprotein recovery, modulators of peptidase, the identification ofpeptidase substrates, binding assays, kit to identify a modulator,Confirmation of identified modulators, large scale screening assays,libraries of test agents, types of test agents, personal care productsand peptidase sequences.

Peptidase and Substantially Homologous Sequences:

The peptidases (or nucleotides encoding them) useful in methodsdescribed herein may be selected from the group consisting of thepeptidase of SEQ ID NO:2 (or SEQ ID NO:1 for the nucleotide encodingit), the peptidase of SEQ ID NO: 4 (or SEQ ID NO:3 for the nucleotideencoding it), and peptidases that are substantially homologous to SEQ IDNO:2 and/or SEQ ID NO:4 (or SEQ ID NO:1 and/SEQ ID NO:3 for thenucleotides encoding them) and remain functional, i.e. the peptidasebinds to a substrate/malodourant precursor as herein-described and/orreacts/cleaves said substrate/malodourant precursor (or in case of thenucleotides they are considered functional if they code for such apeptidase). For example, without limitation, a peptidase or nucleotideencoding a peptidase which is derivable from corynebacteria naturallyoccurring in or isolatable from the human axilla.

A substantially homologous peptidase comprises the following blocks ofconserved partial sequences (the letters refer to the single characteraminoacid code and X is any one of the common aminoacids, and saidaminoacids are numbered starting from the N-terminus of thesubstantially homologous peptidase in its naturally occurring form:

ERDGRWYGRGXADCKG between aminoacid 105 and 150,EGSEEXG between aminoacid 150 and 180,HSGXXGGXAPDA between aminoacid 205 and 255,GGSIPL between aminoacid 385 and 425.

For numbering, either the naturally occurring sequence of thesubstantially homologous peptidase is used, or, before numbering, aswill be apparent to the skilled person, any added tags, fused sequences,or similar are corrected and not considered in the numbering of thesequence so as to number it consistent with the sequence in itsnaturally occurring form, i.e. without such or similar changes that willaffect the numbering.

The conserved sequence blocks are also shown in the figure below.

The identified peptidase enzymes SEQ ID NO:2 and SEQ ID NO:4 wereisolated from Corynebacterium sp. Ax20 (SEQ ID NO:2) and Corynebacteriumjeikeium (SEQ ID NO:4), more particularly from Corynebacterium sp. Ax20,DSM 14267, most closely related to Corynebacterium striatum or glaucum,which has been submitted on the 26, April 2001 to the InternationalDepository Authority DSMZ-German Collection of Microorganisms and CellCultures, D-38124 Braunschweig (Accession Number DSM 14267), andCorynebacterium jeikeium K411 (Tauch et al. 2005, J. Bacteriol. 187(13):4671-82).

Ax20 (DSM 14267) was isolated from human axilla and is most closelyrelated to Corynebacterium striatum according biochemical tests (APICoryne test kit, BioMerieux, France), based on sequence analysis of thefull 16S rRNA gene, it is most closely related to type strains from thespecies Corynebacterium glaucum.

Corynebacterium jeikeium is known for opportunistic infections,especially in immunocompromised patients. K411 was isolated from thehuman axilla. It is a lipid-requiring and multidrug-resistant bacterialspecies of the human skin flora that has been recognised as a nosocomialpathogen.

The term peptidase or peptidases as used herein may refer to the Ax20peptidase (SEQ ID NO:2), the K411 peptidase (SEQ ID NO:4), or peptidasessubstantially homologous thereto as described herein.

The peptidases were classified as metallopeptidases insofar as theyrequire the presence of Zn ions as a cofactors. They are functionallyrelated to the previously identified metallopeptidase (AMRE) thatreleases unsaturated or hydroxylated acid malodourants, and is alsozinc-dependent. The nucleotide sequences encoding the peptidases havebeen isolated and sequenced (SEQ ID NO:1, SEQ ID NO:3). Each peptidasegene sequence can be introduced in a suitable expression vector and usedto produce the peptidase by heterologous expression in the desiredmicroorganism or cells.

Sequences SEQ ID NO: 1 and 2 (from Ax20) were not previously described,and the isolated peptidase of SEQ ID NO:4 has not been previouslydescribed.

In vivo, the peptidases occur intracellularly and can be released fromthe cells by mechanical disruption of the cell envelope. Thus, thepeptidase may be isolated from cellular extracts, in particular cellularextracts obtained from cells derived from organisms selected from thegroup consisting of wild-type corynebacteria strains.

The corynebacteria include, for example, without limitation,Corynebacteria sp., Corynebacterium striatum, Corynebacterium glaucum,Corynebacterium jeikeium, Corynebacterium sp. Ax 20, Corynebacteriumjeikeium K411, Corynebacterium xerosis, Corynebacterium appendicis,Corynebacterium coyleae, Corynebacterium mucifaciens, Corynebacteriumriegelii, and Corynebacterium tuberculostearicum.

A good source of wild-type corynebacteria strains is the human axilla,from which bacterial strains may be cultured and isolated, as is wellknown in the art.

All of the above corynebacteria were isolated from or occur in the humanaxilla, express malodour forming enzymes and are able to form malodour.

Peptidase may be used in form of intact cells, or in isolated form, forexample as a crude extract, or in purified form. For example, a crudeextract may be formed by mechanical disruption of the cells. Optionallypeptidase may be purified. Alternatively peptidase may be formedrecombinantly as described herein.

A substantially homologous peptidase protein includes such proteinswhere the substrate binding and/or catalytic part is replaced with therelevant part of an allelic variant from a different species, forexample another human axilla derived bacterium including but not limitedto bacteria of the genus Corynebacterium.

Further, substantially homologous peptidase nucleotide or polypeptidesequences may be formed by conservative mutations and/or point mutationsand include any conservatively modified variant as detailed below.

With respect to nucleotide sequences, conservatively modified variantsmeans nucleotides which encode identical or essentially identical aminoacid sequences (conservatively substituted amino acids, i.e. lysineswitched to arginine and further examples as explained herein-below).

Because of the degeneracy of the genetic code, a large number ofnucleotides different in sequence but functionally identical encode anygiven polypeptide/protein. Such nucleotide variations are “silentvariations,” which are one species of conservatively modifiedvariations. Each nucleotide sequence which encodes a polypeptide alsodescribes every possible silent variation of the nucleotide. Therefore,each codon in a nucleotide (except AUG, which is ordinarily the onlycodon for methionine, and TGG, which is ordinarily the only codon fortryptophan) can be modified to yield a functionally identical nucleotidesequence that will produce an identical polypeptide. Accordingly, eachsilent variation of a nucleotide which encodes a polypeptide is implicitin each given nucleotide sequence.

With respect to amino acid sequences, amino acid substitutions may beintroduced using known protocols of recombinant gene technologyincluding PCR, gene cloning, site-directed mutagenesis of cDNA,transfection of host cells, and in-vitro transcription which may be usedto introduce such changes to the peptidase sequence. The variants canthen be screened for peptidase activity. Conservative substitutiontables providing functionally similar amino acids are well known in theart. For example, one exemplary guideline to select conservativesubstitutions includes (original residue followed by exemplarysubstitution): ala/gly or ser; arg/lys; asn/gln or his; asp/glu;cys/ser; gln/asn; gly/asp; gly/ala or pro; his/asn or gln; ile/leu orval; leu/ile or val; lys/arg or gin or glu; met/leu or tyr or ile;phe/met or leu or tyr; ser/thr; thr/ser; trp/tyr; tyr/trp or phe;val/ile or leu.

An alternative exemplary guideline uses the following six groups, eachcontaining amino acids that are conservative substitutions for oneanother: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid(D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine(R), Lysine (1); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine(V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

Another alternative guideline is to allow for all charged amino acids asconservative substitutions for each other whether they are positive ornegative.

In addition, individual substitutions, deletions or additions thatalter, add or delete a single amino acid or a small percentage (forexample up to 26%, or up to 20%, or up to 10%) of amino acids in anencoded sequence are also considered to be conservatively modifiedvariations.

Substantially homologous nucleotide or polypeptide sequences have thedegree of sequence identity indicated below while retaining thecatalytic activity of the polypeptide on the substrates as definedherein.

% Sequence identity:

A substantially homologous nucleotide sequence has a % sequence identityof at least at least 50%, at least 55%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%, or at least 98%.

A substantially homologous polypeptide sequence has a % sequenceidentity of at least at least 40%, at least 42%, at least 50%, at least60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least98%

Notably, when comparing the peptidase Ax20 (SEQ ID NO:1 and NO:2) andpeptidase K411 (SEQ ID NO:3 and NO:4), which both have the samecatalytic activity on the same physiological substrate, based on theiraminoacid sequence, their identity is 42%, and based on their nucleotidesequences, their identity is 55%.

Calculation of % Sequence Identity is determined as follows.

BLAST (Basic Local Alignment Search Tool) is the heuristic searchalgorithm employed by the programs blastn which is available athttp://www.ncbi.nlm.nih.gov.

To determine % identity of a nucleotide query sequence against anothernucleotide sequence, Blastn is used, using default parameters of BLASTversion 2.2.1.3, including an EXPECT (statistical significance thresholdfor reporting matches against database sequences) of 10, and DUSTfiltering.

To determine % identity of a polypeptide query sequence against anotherpolypeptide sequence, Blastp is used, using default parameters of BLASTversion 2.2.1.3, including an EXPECT of 10, and DUST filtering.

Substantially homologous nucleotide sequences include, withoutlimitation, sequences that are selectively hybridising to one or more ofthe peptidase nucleotide sequences described herein, or to theircomplement, under stringent hybridisation conditions detailed below.Stringent conditions are temperature of 42° C. in a solution consistingof 50% formamide, 5×SSC, and 1% SDS and washing at 65° C. in a solutionconsisting of 0.2×SSC and 0.1% SDS (1×SSC=0.15 M NaCl, 0.015 M Na3Citrate pH 7.0).

Background hybridisation may occur because of other nucleotide sequencespresent, for example, in the genomic DNA library being screened.

A signal that is 2 fold less intense or optionally 10 fold less intensethan the specific interaction observed with the target DNA is consideredbackground. The intensity of interaction may be measured, for example,by radiolabeling the probe, e.g. with 32P.

The term “isolated” as used herein means taken from the environment thatsomething originated from and transferred to a different environment,including the environment of an intact cell and transferring it to acell lysate.

By “purified form”, is meant more than 80%, for example, withoutlimitation, more than 90%, more than 95%, more than 98%, more than 99%or more with respect to other protein and/or nucleic acid contaminants(w/w).

Substrates of Peptidase

The substrates of the peptidase (for example, without limitation,precursor I) may generally be described as dipeptide derivatives.Examples of substrates include, without limitation, simple dipeptidessuch as ala-ala and ala-gly. The substrates with the highest affinity tothe peptidase are cys-gly and cys-ala derivatives, in particularL-cys-L-gly and L-cys-L-ala derivatives wherein the S atom of thecys-gly or cys-ala residue is alkylated as described herein.

The cys-gly derivatives can be the naturally occurring, for example,without limitation, precursor I, or synthetic analogues including butnot limited to S-benzyl-Cys-Gly.

Whether a given dipeptide derivative is a peptidase substrate may beeasily determined by incubating it with the SEQ ID NO: 2 or SEQ ID NO:4peptidase and determining whether said substrate is enzymaticallyreacted by the peptidase as described herein.

Substrates include, without limitation, substrates with high affinity tothe enzyme which may be of formula FI shown below

and wherein X is selected from the group consisting of S and O, andwherein R1 is a residue selected from the group consisting of H andmethyl, and wherein R2 is a residue selected from the group consistingof a straight chain or branched C1 to C10 alkyl, a straight chain orbranched C1 to C10 alkanol, a phenyl, and a benzyl.

A particular group of useful substrates are the substrates of formula FIwherein X is S, R1 is methyl, and R2 is a residue selected from thegroup consisting of a straight chain or branched C1 to C10 alkyl, astraight chain or branched C1 to C10 alkanol, a phenyl, and a benzyl.

With regard to R2 residues, “C1 to C10 alkyl” includes, withoutlimitation, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octly,nonyl, and decyl; and “C1 to C10 alkanol” includes, without limitation,methanol, ethanol, propanol, butanol, pentanol, hexanol, heptanol,octanol, nonanol, decanol, and optionally any branched forms of alkyl oralkanol.

Physiologically relevant substrates are those indicated herein-above as“precursor I” and have the general formula FII

wherein R1 is an alkyl residue selected from the group consisting ofmethyl, ethyl, propyl, butyl, pentyl, and hexyl,and wherein R2 and R3 are independently selected from the groupconsisting of H and methyl.

Particular examples of peptidase substrates include, without limitation,S-benzyl-Cys-Gly, O-benzyl-Ser-Gly,(1-(2-hydroxyethyl)-1-methylbutyl)-L-cysteinyl-glycine,S-benzyl-Cys-Ala, O-benzyl-Ser-Ala, Pro-Gly, Ala-Gly, Ala-Ala, Pro-Ala.Their chemical structures are shown in the table below.

S-benzyl-Cys-Gly

O-benzyl-Ser-Gly

(1-(2-hydroxyethyl)-1- methylbutyl)-L- cysteinyl-glycine

S-benzyl-Cys-Ala

O-benzyl-Ser-Ala

Pro-Gly

Ala-Gly

Ala-Ala

Pro-Ala

Peptidase Reaction Products:

The products of an enzymatic reaction with peptidase are two free aminoacids for the simple dipeptide substrates or for gly derivatives, a freeglycine and a S-substituted cys or an O-substituted ser derivative, orfor ala derivatives, a free alanine and a S-substituted cys or anO-substituted ser derivative.

The final malodourant product is only formed when the resultingenzymatic reaction product of peptidase (for example, precursor II) isexposed to a β-lyase by which it is cleaved to release asulfanylalkanol.

The novel constructs and products that are provided (DNA, vectors,recombinant bacteria, protein/peptidase enzyme) are useful whenscreening, without limitation, for inhibitors of the peptidase reaction.

Identification of Modulators and Inhibitors:

Modulators which are inhibitors are of interest to add to products (inparticular to personal care products including but not limited todeodorants) to prevent the formation of body malodour. Similarly, thescreening methods described herein below could be used to identify othermodulators such as enhancers, which are to be avoided in such products.

To identify a modulator, the peptidase and its substrate are exposed toat least one test agent (potential modulator) in a suitableconcentration, for example, without limitation, from 1 nm to 1 mM, orfrom 10 nm to 10 microM.

Then the effect of the modulator on the enzymatic cleavage/reaction rateis monitored by methods well known in the art, for example, withoutlimitation, by methods as described herein.

A change of the enzymatic cleavage/reaction rate is determined by theeduct cleavage rate or product formation rate (the latter includessecondary products).

For example, the cleavage/reaction rate is determined by monitoring therate that the substrate disappears, or the rate that cleavage productsof the substrate appear (for example, the rate that the free aminoacidreaction product appears, including but not limited to gly or ala).

In one embodiment of a high-throughput screen of potential modulators,the release of free L-gly or L-ala is measured by derivatising the freeNa group with an amine-group derivatising agent, which upon reactionwith the amine group forms a chromophore or a fluorescent molecule.Useful in this regard may be the use of fluorescamine (Fluka, Buchs,Switzerland) to form a fluorescent molecule upon reaction with L-gly orL-ala. Finally, the cleavage of the L-gly-substrate may be compared tocontrol reactions and the potential of the test compounds to influence,in particular to inhibit, the reaction may thereby be quantified.

If the β-lyase or another enzyme acting on the peptidase reactionproduct is added to the reaction as well, then alternatively oradditionally, the rate by which the malodourant (sulfanylalcohol) orother released sulfur molecules appear can be monitored. A change in themonitored rate indicates the effect of a test agent and potentialmodulator on the enzyme. An inhibitor is identified by a lower rate ofcleavage product formation or lower rate of free amino acid release orlower rate of sulfur molecule formation when compared to the reaction inabsence of the inhibitor test agent or a pre-determined standard rate.

The cleavage/reaction rates can be monitored, for example, withoutlimitation, by the following methods: analysis of the substrate or theformed free amino acids or other cleavage products by high performanceliquid chromatography (HPLC) or thin layer chromatography (TLC) orcapillary electrophoresis; fluorescence spectrophotometry after reactingthe free amino acid with a fluorescent probe (for example, withoutlimitation, fluorescamine); fluorescence spectrophotometry afterreacting the free sulfur molecule with a fluorescent probe (for example,without limitation, monobromobimane); gas chromatography of releasedsulfur molecules; or the detection of reaction products by biochemicaltests.

Useful peptidases include, without limitation, the peptidase of SEQ IDNO:2 and the peptidase of SEQ ID NO:4. The peptidase of SEQ ID NO:4 hasa lower activity then the peptidase of SEQ ID NO:2 but is still veryuseful as a screening target or to verify screening hits and/or toscreen for a broad range activity of inhibitors in different bacterialspecies.

These screens can be performed subsequently or in parallel, using bothenzymes in one reaction well.

In parallel or subsequently to the screening methods employing peptidaseas described herein, additional malodour-forming enzymes can be used,for example, without limitation, one or more of β-lyase and AMRE. Tothat end, β-lyase and/or AMRE may be incubated with the peptidase andits substrate and the test agent in parallel or subsequently and theeffect of the test agent on the cleavage/reaction rate of the enzymes isdetermined by the change in formation of a sulfanylalkanol (released bypeptidase and 3-lyase) and/or carboxylic acids or glutamine (released byAMRE). Alternatively, the change in the reaction rate of the educts ofthe reaction may be determined.

Identified inhibitors of one or more of peptidase SEQ ID NO:2, peptidaseSEQ ID NO:4, and substantial homologs are desirable deodorantingredients since they inhibit the rate limiting step in releasingsulfur chemicals from odourless sweat. Particularly interesting areinhibitors which inhibit more than one corynebacterial peptidase, forexample, without limitation, both peptidase SEQ ID NO:2 and peptidaseSEQ ID NO:4, and therefore will provide a broad band activity against anumber of different corynebacteria species.

The results in the examples herein-below show that β-lyase cleaves onlythe cys-conjugate but not precursor I of FIII, indicating that theβ-lyase alone cannot be responsible for the cleavage of thequantitatively most abundant substrate in the axilla secretion. Incontrast, the peptidase-containing extract from C. sp. Ax 20 isolatedfrom the axilla of a human test subject does release sulfanylalkanolmalodourants from precursor I of FIII (in the examples: cys-glyconjugate) and this reaction is stopped by the metallopeptidaseinhibitor o-phenantrolin. This is surprising since WO 2006079934postulates a β-lyase cleaving precursor I of FIII. It follows that thecell extracts contain another enzyme than a β-lyase that is involved inthe cleavage of precursor I of FIII. But since no single fraction of thecell extract of Ax20 can release the malodourant sulfur volatile fromprecursor I of FIII as shown in the examples herein and since a singlefraction in presence of beta-Lyase can release the malodourant sulfurvolatile from precursor I of FIII, again, as shown in the examplesherein, it follows, that two enzymes sequentially release themalodourant from precursor I of FIII, namely a peptidase releasing gly,and thereby forming the substrate for the beta-lyase which in turnreleases the sulfur molecule.

Cells Used in the Assays:

Suitable bacterial cells include all corynebacteria that naturallyexpress peptidase, for example, without limitation, Corynebacterium sp.,Corynebacterium sp. Ax20, Corynebacterium striatum, Corynebacteriumglaucum, Corynebacterium jeikeium, Corynebacterium jeikeium K411,Corynebacterium xerosis, Corynebacterium appendicis, Corynebacteriumcoyleae, Corynebacterium mucifaciens, Corynebacterium riegelii, andCorynebacterium tuberculostearicum.

Alternatively the peptidase may be heterologously expressed in a hoststrain, for example, without limitation, a bacterial strain (including,without limitation, an E. coli strain), a yeast strain, or an eukaryoticcell line (including, without limitation, insect cells, mammalian cells,amphibian cells, and worm cells). These host strains are transformedwith a suitable vector carrying the nucleotide sequence coding for thepeptidase and the relevant control elements for the host, as is wellknown in the art.

The vector constructs for expressing the peptidase may be produced in amanner known per se using polymerase chain reaction (PCR) to amplify thecoding region from chromosomal DNA of a corynebacterium and, afterverification of the sequence, sub-cloning the coding sequence into asuitable vector. Suitable vectors are commercially available, forexample vectors from Invitrogen (Groningen, The Netherlands). A usefulvector is the vector pET-3a (Studier and Moffatt, 1986). The resultingplasmids are transformed into a suitable E. coli host strain.Suitability of a vector-host strain combination depends on the chosenhost-strain for which a vector is compatible with or optimized for (forexample the vector pET-3a is transformed into the host strainBL21(DE3)).

Alternatively, a variety of expression vector/host systems can be usedto contain and express sequences encoding the peptidase. These include,for example, different microorganisms including bacteria transformedwith recombinant bacteriophage, plasmid, or cosmid DNA expressionvectors; yeast transformed with yeast expression vectors; insect cellsystems infected with viral expression vectors (for examplebaculovirus), or with bacterial expression vectors (for example pBR322plasmids).

Yeast expression systems may be used for production of peptidase. Anumber of vectors containing constitutive or inducible promoters, suchas alpha factor, alcohol oxidase, and PGH promoters, may be used in theyeast Saccharomyces cerevisiae or Pichia pastoris. In addition, suchvectors direct either the secretion or intracellular retention ofexpressed proteins and enable integration of foreign sequences into thehost genome for stable propagation.

For the expression of heterologous proteins in insect cell lines is, forexample, derivatives of the Lepidopteran baculovirus, Autographacalifornica multicapsid nucleo-virus (AcMNPV) can be used. In thissystem, foreign gene expression is directed by a very strong late viralpromoter, either the polyhedrin or p10 promoters, and a wide array ofvectors is available that optimises expression and recovery ofrecombinant proteins. These vectors enable expression of bothmembrane-bound and secreted proteins at high levels. A number of vectorsare commercially available, for example the InsectSelect™ System fromInvitrogen.

Assays Using Purified Peptidase:

Alternatively to using a cell-based assay, it usually will be simpler topurify the peptidase from peptidase expressing cells describedherein-above using methods well known in the art and subsequentlyperforming an enzymatic assay contacting the purified peptidase, itssubstrate and a test agent in vitro and determining a change in thepeptidase reaction rate as described herein. Optionally, the results maybe validated in vivo using corynebacteria derivable from the humanaxilla as described herein.

Peptidase Transcription Assays:

Alternatively to cell-based or enzymatic assays, an assay to identifymodulators that act on the level of gene transcription may be performed.Test agents are added to peptidase expressing wildtype corynebacteria.After a finite time (for example, 30 min to 8 h) the test agents areremoved by washing the bacterial cells or by harvesting the cells bycentrifugation. The bacterial cells are then analyzed for the amount ofthe peptidase, and this value is compared to control cells not exposedto the test agent. Inhibitors reduce the amount of peptidase formed bythe bacteria and therefore their potential to form malodour. The amountof peptidase of the bacterial cells can be determined either by anactivity assay using the methods and substrates as described herein orby raising a specific antibody (either a monoclonal antibody or apolyclonal antibody) to a peptidase including, without limitation, SEQID 2 or SEQ ID 4 and then using a suitable immunological detectionmethod, for example, without limitation, immuno dot-blot, western-blot,or enzyme-linked immunosorbent assay (ELISA), to specifically detect thepeptidase in the bacterial cells.

Expression Systems for Peptidase:

In order to express cDNAs encoding the desired proteins, one typicallysubclones the appropriate cDNA into an expression vector that contains astrong promoter to direct transcription, a transcription/translationterminator, and a ribosome-binding site for translational initiation.Suitable bacterial promoters are well known in the art, for example, E.coli, Bacillus sp., and Salmonella, and kits for such expression systemsare commercially available. Similarly, eukaryotic expression systems formammalian cells, yeast, and insect cells are commercially available. Theeukaryotic expression vector may be, for example, an adenoviral vector,an adeno-associated vector, or a retroviral vector.

An expression cassette should also contain a transcription terminationregion downstream of the structural gene to provide for efficienttermination. The termination region may be obtained from the same geneas the promoter sequence or may be obtained from different genes.

For expression of the proteins, conventional vectors for expression ineucaryotic or procaryotic cells well known in the art may be used.Examples of vectors include bacterial expression vectors, for example,plasmids including pBR322-based plasmids, pBAD base plasmid, pSKF, andpET23D, and fusion expression systems, for example, GST and LacZ.

Expression vectors containing regulatory elements from eukaryoticviruses are typically used in eukaryotic expression vectors, for exampleSV40 vectors, cytomegalovirus vectors, papilloma virus vectors, andvectors derived from Epstein-Barr virus. Other exemplary eukaryoticvectors include pMSG, pAV009/A⁺, pMTO10/A⁺, pMAMneo-5, baculoviruspDSVE, pcDNA3.1, pIRES and any other vector allowing expression ofproteins under the direction of the SV40 early promoter, SV40 laterpromoter, metallothionein promoter, murine mammary tumor virus promoter,Rous sarcoma virus promoter, polyhedrin promoter, or other promotersshown effective for expression in eukaryotic cells.

Some expression systems have markers that provide gene amplificationsuch as thymidine kinase, hygromycin B phosphotransferase, dihydrofolatereductase and the like. The elements that are typically included inexpression vectors may also include a replicon that functions in E.coli, a gene encoding drug resistance to permit selection of bacteriathat harbor recombinant plasmids, and unique restriction sites innon-essential regions of the plasmid to allow insertion of eukaryoticsequences. The particular drug resistance gene chosen is not critical,any of the many drug resistance genes known in the art are suitable. Theprokaryotic sequences are optionally chosen such that they do notinterfere with the replication of the DNA in eukaryotic cells, ifnecessary.

In bacterial systems the peptidase cDNA fragment may be expressed aloneor as a fusion protein wherein the peptidase protein of interest isfused to the E. coli periplasmic maltose-binding protein (MBP) whereinthe MBP, including its signal peptide, is linked to the amino terminusof the peptidase. The wild-type peptidase cDNA or the MBP: peptidasefusion cDNA is subcloned into a suitable plasmid, for example pBR322,where in E. coli, peptidase expression is driven by the lac wild-typepromoter.

Particular examples of vector-host strain combinations are the vectorpPROTet.E133 in strain E. coli DH5αPRO (Clontech, Palo Alto, Calif.,USA) or the vector pBAD-myc-his-A in E. coli strain TOP 10 (Invitrogen,Groningen, The Netherlands).

Further examples of expression vectors and host strains are described inT. Maniatis et al. (Molecular Cloning, cold spring Harbor Laboratory,1982).

In vitro transcription and translation is another alternative to expresspeptidase.

Overexpression of Peptidase:

Peptidase may be overexpressed by placing it under the control of astrong constitutive promoter, for example the CMV early promoter.Alternatively, overexpression may be achieved under control of aninducible promoter, for example, without limitation, the arabinosepromoter in the vector pBAD-myc-his-A or the T-rex system describedherein below.

Transfection of Peptidase Expression Vector Constructs into Cells:

Standard transfection methods can be used to produce bacterial,mammalian, yeast or insect cell lines that express large quantities ofthe protein.

Any known method for introducing nucleotide sequences into host cellsmay be used. It is only necessary that the particular geneticengineering procedure used be capable of successfully introducing therelevant genes into the host cell capable of expressing the proteins ofinterest. These methods may involve introducing cloned genomic DNA,cDNA, synthetic DNA or other foreign genetic material into a host celland include the use of calcium phosphate transfection, polybrene,protoplast fusion, electroporation, liposomes, microinjection, plasmavectors, viral vectors and the like.

For example, without limitation, the T-Rex™ expression system(Invitrogen Corp., Carlsbad, Calif.) may be used. The T-Rex™ System is atetracycline-regulated mammalian expression system that uses regulatoryelements from the E. coli Tn10-encoded tetracycline (Tet) resistanceoperon. Tetracycline regulation in the T-Rex™ System is based on thebinding of tetracycline to the Tet repressor and depression of thepromoter controlling expression of the gene of interest.

After transfection, the transfected cells may be cultured using standardculturing conditions well known in the art. It will be apparent to theskilled person that different cells require different culture conditionsincluding appropriate temperature and cell culture media.

Peptidase Protein Recovery:

If desired, the protein may be recovered from the cells using standardtechniques. For example, the cells may be burst open either mechanicallyor by osmotic shock. The resulting crude enzyme may be used as such ormay be separated from one or more of cell debris, cell proteins, cellnucleic acids, and cell contaminants, for example by being subjected toprecipitation and chromatography steps, including but not limited toion-exchange, hydrophobic interaction, reverse-phase, and size exclusionchromatogrphy steps. After each step the eluted protein may be furtherpurified and/or concentrated by methods including but not limited tofiltration and ultrafiltration. Alternatively, the recombinant proteinmay be recovered from the culture medium in which the recombinant cellshad been cultured (provided the peptidase has been expressed togetherwith a sequence that provides for export into the culture medium).

Modulators that May be Identified by the Assays:

Modulators and in particular inhibitors of peptidase activity can beidentified as described herein below.

There now follows a definition of the agents to be identified in themethods described herein.

A modulator is an agent that effects an increase or decrease of one ormore of the following: the binding of a substrate to the peptidase, thereaction of the substrate (for example, without limitation, amalodourant precursor) to the malodourant or a second malodourantprecursor. The modulator can itself bind to the peptidase, either at thesubstrate binding site or elsewhere, and can either be cleaved orreacted at a different rate or not at all, or can bind to the substrateand thereby affect its reaction/cleavage rate.

Modulators include various types of compounds, including smallmolecules, peptides, proteins, nucleotides, antibodies or fragmentsthereof. These can be derived from various sources including syntheticor natural, extracts of natural material, for example from animal,mammalian, insect, plant, bacterial or fungal cell material or culturedcells, or conditioned medium of such cells.

A substrate is an agent that binds to the peptidase and is cleaved orreacted by it.

An inhibitor is a modulator that decreases the binding of a substrate tothe peptidase as compared to the binding of the substrate in the absenceof inhibitor, and/or decreases the reaction/cleavage rate of thesubstrate, and/or decreases overall peptidase activity.

An enhancer increases the binding of a substrate to the peptidase ascompared to the binding of the substrate in the absence of enhancer,and/or increases the reaction/cleavage rate of the substrate, and/orincreases overall peptidase activity.

The activity, or changes in activity, of a peptidase binding a substrateand reacting/cleaving it to form a second precursor and finally themalodourant can be determined by the methods described herein-below.

Identification of Substrates:

To identify a substrate, a test agent is incubated with peptidase andthe formation of reaction products or the disappearance of the potentialsubstrate is followed by analytical methods as described herein. Anycompound which is reacted by the peptidase is defined as a substrate. Torate the affinity of a substrate versus other known substrates, dilutionseries of the substrate are incubated with a fixed concentration ofpeptidase, and the cleavage rate at each substrate concentration after agiven time is determined. Based on the resulting curve, the biochemicalparameters v_(max) (maximal reaction rate in molecules substratescleaved per second by one molecule of peptidase) and K_(m) (Michaelisconstant giving the substrate concentration where the enyme is activewith 50% of maximal reaction rate) are determined. A low K_(m) indicatesa high affinity of the peptidase for its substrate and thus a substratewith a low K_(m) and a high v_(max) is a particularly good substrate forthe enzyme. However, for screening assays, substrates with a high Km andlow v_(max) may also be used.

Kit to Identify a Modulator:

A kit, for example a screening kit or high throughput screening kit,that comprises isolated peptidase (from wild-type or recombinant cells)or recombinant cells that express the peptidase, or a substantiallyhomologous sequence thereto; and that comprises a substrate of thepeptidase, for example, without limitation, S-benzyl-Cys-Gly,O-benzyl-Ser-Gly,(1-(2-hydroxyethyl)-1-methylbutyl)-L-cysteinyl-glycine,S-benzyl-Cys-Ala, O-benzyl-Ser-Ala, Pro-Gly, Ala-Gly, Ala-Ala, Pro-Ala.

The substrate is provided in suitable concentrations, for example 1microM to 10 mM, or 10 microM to 1 mM, for example 50 microM to 1 mM, or50 microM to 500 microM.

Optional kit components may include a suitable medium for culturing therecombinant cells provided, and a culture device to grow the cells in,for example, a microtiter plate, these optional components will bereadily available to the skilled person.

The kit may be used as follows:

(i) growing recombinant cells that express the peptidase, oralternatively, providing an isolated peptidase,(ii) adding at least one test agent in the presence of a peptidasesubstrate in a suitable concentration, and(iii) determining a change in cleavage/reaction rate of thesubstrate/its product(s) by the peptidase or a change in substratebinding to the peptidase by comparing the response in presence andabsence of the test agent, and the test agent is thereby identified as amodulator.

In particular, for the kit using isolated peptidase:

(i) test agents, optionally at concentrations from about 1 nanomolar toabout 5 millimolar, are added to a peptidase containing assay buffer,(ii) after a defined pre-incubation period which allows test agentbinding to the peptidase (optionally, 0 to 15 minutes), the selectedsubstrate is added in a suitable concentration,(iii) a change in cleavage/reaction rate of the substrate or itsproduct(s) by the peptidase or a change in substrate binding to thepeptidase is determined by comparing the response in presence andabsence of the test agent, and the test agent is thereby identified as amodulator.

A similar assay can be performed where the peptidase is not in anisolated form but in the form of a cellular extract or is provided inform of intact prokaryotic or eucaryotic cells:

(i) recombinant cells that express the peptidase protein are grown inculture,(ii) test agents, optionally at concentrations from about 1 nanomolar to5 millimolar, are added to the culture medium in the presence of thesubstrate in a suitable concentration,(iii) a change in cleavage reaction rate of the substrate or itsproduct(s) by the peptidase or a change in substrate binding to thepeptidase is determined by comparing the response in presence andabsence of the test agent, and the test agent is thereby identified as amodulator.

For example, without limitation, step (iii) may be performed accordingto any one of the methods described herein above. This may requirespecifically chosen or adapted recombinant cells, which are alsodescribed herein-above.

Confirmation of Identified Modulators:

A modulator identified by a method described herein-above may easily beconfirmed by simple sensory experiments using a panel of test persons tosmell samples as detailed below. The samples have been exposed to apeptidase and a β-lyase and contain as a substrate a compound of formulaFI below and any formed enzymatic reaction products.

wherein X is selected from S and wherein R1 is residue selected from thegroup consisting of H and methyl and wherein R2 is a residue selectedfrom the group consisting of a C1 to C10 alkyl, C1 to 010 alkanol, aphenyl, and a benzyl.

The samples are smelled by the panel in comparison to a negative controlwithout modulator to confirm a modulator that modulates, for exampleinhibits, malodour formation.

Large Scale Screening Assays:

The assays described herein-above are well suited for screeninglibraries for agents that modulate peptidase activity.

The assays may be designed to screen large chemical libraries byautomating the assay steps and providing compounds from any convenientsource to the assays, which are typically run in parallel (for examplein microtiter formats on microtiter plates in robotic assays).

Assays may be run in high throughput screening methods that involveproviding a combinatorial chemical or peptide library containing a largenumber of potential modulators. Such libraries are then screened in oneor more assays described herein-above to identify those library agents(particular chemical species or subclasses) that display the activitydescribed herein-above. The modulators thus identified can be directlyused or may serve as leads to identify further modulators by making andtesting derivatives.

Synthetic compound libraries are commercially available from a number ofcompanies including Maybridge Chemical Co. (Trevillet, Cornwall, UK),Comgenex (Princeton, N.J.), Brandon Associates (Merrimack, N.H.), andMicrosource (New Milford, Conn.).

Binding Assays:

Alternatively to the functional methods described herein-above thatmeasure a change in cleavage/reaction rate by measuring increase ordecrease of educts and direct or indirect products (for example, withoutlimitation, substrate/precursor I, precursor II, malodourant), substratebinding may be determined by binding assays that measure the binding ofa substrate to the peptidase, which are well known in the art. Bindingof a modulator to a peptidase polypeptide can be determined, forexample, without limitation, by measuring changes in spectroscopiccharacteristics (for example fluorescence, absorbance, or refractiveindex), hydrodynamic methods (employing for example shape),chromatography, measuring solubility properties of a peptidasepolypeptide.

Libraries of Test Agents:

A combinatorial chemical library is a collection of diverse chemicalcompounds generated by either chemical synthesis or biologicalsynthesis, by combining a number of chemical “building blocks” such asreagents. For example, a linear combinatorial chemical library such as apolypeptide library is formed by combining a set of chemical buildingblocks (amino acids) in every possible way for a given compound length(i.e., the number of amino acids in a polypeptide compound). Millions ofchemical compounds can be synthesized through such combinatorial mixingof chemical building blocks.

A rare chemical library is available from Aldrich (Milwaukee, Wis.).

Libraries of natural compounds in the form of bacterial, fungal, plantand animal extracts are commercially available for example from PanLaboratories (Bothell, Wash.) or MycoSearch (NC), or are readilyproduceable by methods well known in the art. Additionally, natural andsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical, and biochemical means.

Other libraries include protein/expression libraries, cDNA librariesfrom natural sources, including, for example, foods, plants, animals,bacteria, libraries expressing randomly or systematically mutatedvariants of one or more polypeptides, genomic libraries in viral vectorsthat are used to express the mRNA content of one cell or tissue.

In a high throughput assay, it is possible to screen up to severalthousand different modulators or substrates in a single day. Inparticular, each well of a microtiter plate can be used to run aseparate assay against a selected potential modulator/substrate, or, ifconcentration or incubation time effects are to be observed, every 5-10wells can test a single modulator. Thus, a single standard microtiterplate can assay about 100 modulators. If 1536 well plates are used, thena single plate can easily assay from about 100 to about 1500 differentcompounds. It is possible to assay several different plates per day;assay screens for up to about 6,000-20,000 different compounds ispossible.

Types of Test Agents that May be Tested for their Peptidase ModulatingEffect in the Assay Methods:

The test agents may be any agent including small chemical compounds,chemical polymers, biological polymers, peptides, proteins, sugars,carbohydrates, nucleic acids and lipids. An agent can be a syntheticcompound, a mixture of compounds, a natural product or natural sample,for example plant extract, culture supernatant, or tissue sample.

Personal Care Products:

The identified enzyme inhibitors/malodour counteractants may be added tovarious products to prevent body malodour. For example, personal careproducts include, without limitation, deodorant, antiperspirant, lotion,cream, salve, powder, body lotion, unguent, soap, shampoo, finefragrance, eau de Cologne, eau de toilet. Personal care products may beperfumed or may be perfume-free. Such products usually contain a numberof excipients that are well known in the art. The malodourcounteractanteffect of personal care products can be further improved by leaving outany ingredients indentified as enhancers of peptidase.

The identified modulator/inhibitor may be added to an aqueous solution,emulsion, alcoholic solution, silicon solution, oil or wax.

Sequences of Nucleic Acids and Proteins:

The sequences employed in the constructs and methods described-hereincan be found in the sequence listing herein-below.

SEQ ID NO: 1: Nucleotide sequence of peptidase from Corynebacterium sp.Ax20

SEQ ID NO: 2: Aminoacid sequence of peptidase from Corynebacterium sp.Ax20

SEQ ID NO: 3: Nucleotide sequence of peptidase from Corynebacteriumjeikeium K411

SEQ ID NO: 4: Aminoacid sequence of peptidase from Corynebacteriumjeikeium K411

SEQ ID NO:5: Nucleotide sequence of β-lyase from Corynebacterium sp.Ax20

SEQ ID NO:6: Aminoacid sequence of β-lyase from Corynebacterium sp. Ax20

There now follows a series of examples that serve to illustrate theabove-described subject matter. The following examples are merelyillustrative and should not be construed as limiting the subject matterdescribed herein including polypeptides, nucleic acids/nucleotides,expression vectors, host cells, methods, kit and personal care productsin any manner.

EXAMPLES

All examples use the DNA sequences derived from Corynebacteria, inparticular Corynebacterium sp. Ax20 (“Ax20”), DSM 14267, which has beensubmitted on the 26, April 2001 to the International DepositoryAuthority DSMZ-German Collection of Microorganisms and Cell Cultures,D-38124 Braunschweig (Accession Number DSM 14267), and Corynebacteriumjeikeium K411 (“K411”) (Tauch et al. 2005, J. Bacteriol. 187(13):4671-82).

In the examples, the cys-gly-conjugate refers to the following dipeptidecompound:

The cys-conjugate in the examples refers to the following compound:

“β-lyase” in the examples refers to the β-lyase enzyme described inNatsch et al. 2004, Chemistry & Biodiversity, 1, 1058. The full openreading frame of the β-lyase gene (metC gene, 1134 bp) is available fromGenebank under the accession number AY646680(gi|51556860|gb|AY646680.1|[51556860]). It is also included in thesequence listing (SEQ ID NO:5, nucleotide sequence, and SEQ ID NO:6,protein sequence).

“AMRE” or “AMRE enzyme” is short for “axilliary malodour releasingenzyme” and designates the malodourous acid releasing enzymeNa-acyl-glutamine-aminoacylase described by Natsch in EP1258531. TheAMRE aminoacid sequence is SEQ ID NO: 1 in EP 1258531, the open readingframe of the AMRE gene is SEQ ID NO:5 in EP1258531. It's heterologousexpression is described in example 6 of EP1258531

Example 1 Isolation of Peptidase (SEQ ID NO:2)

A cellular extract was formed as follows: an overnight culture of Ax20was harvested by centrifugation and resuspended in a small volume ofBuffer A (50 mM NaCl; 50 mM NaH2PO4/K2HPO4; pH 7), amended with a10-fold volume of glass beads (425-600 μm, Sigma, St-Louis, USA) andmechanically disrupted by vortexing them at maximal speed for 30 min.The lysates were centrifugated and the supernatants were saved. Theresulting Corynebacterium sp. Ax20 cellular extract was thensubsequently fractionated over the following columns: 1.Phenyl-sepharose (hydrophobic interaction chromatography), 2. Mono-Q(strong anion exchange), 3. Mono-P (weak anion exchange) and 4. Superdex200 (gel filtration).

Samples of each fraction were incubated both with the β-lyase and withthe cys-gly-conjugate at the same time, and the release of3-sulfanyl-3-methyl-hexanol was determined by a fluorescence testspecific for free SH groups: To this end the enzyme reaction was stoppedby adding 0.5 volumes of NaCO3-buffer (0.1 M, pH 8.8) containing 0.5 mMmonobromobimane, thus derivatising the free thiol-group. Fluorescencewas then measured on a Flex-station (Molecular devices, Sunnyvale,Calif., USA) with an excitation wavelength of 385 nm and an emissionwavelength of 480 nm.

A single peak of activity (3-sulfanyl-3-methyl-hexanol release) wasfound in each purification step, indicating that only one enzyme isinvolved in the release reaction.

After these four purification steps, the preparation was separated on aSDS-Page-gel. The gel band unique to the active fractions was excisedand the peptidase thereby obtained.

Example 2 Cloning of the Gene (SEQ ID NO:1) and Isolation of the NucleicAcid Sequence Coding for the Peptidase (SEQ ID NO:2)

The peptidase obtained in example 1 was submitted to a tryptic digestand peptide sequence analysis. Based on these isolated peptidessequences, primers were designed to amplify a partial sequence of thegene by polymerase chain reaction using genomic DNA of Corynebacteriumsp. Ax20 as a template.

The complete gene sequence was then isolated by chromosomal walking witha chromosomal library of Corynebacterium sp. Ax20.

Thereby the complete open reading frame of the peptidase sequence wasobtained as shown in SEQ ID NO:1.

Example 3 Heterologous Expression, Production and Purification of theAx20 Peptidase

The open reading frame (ORF) of the peptidase (SEQ ID NO:1) wasamplified from chromosomal DNA of Corynebacterium sp. Ax 20 by PCR usingspecific primers. The ORF was ligated to a sequence coding for a 6×Histidine-Tag and cloned into an expression vector (pET-3a, Studier andMoffatt, 1986). The resulting plasmids were transformed into an E. colistrain (BL21(DE3)).

Alternatively, the ORF without the 6× Histidine-Tag was cloned into thesame vector.

The recombinant E. coli strains with or without 6× Histidine-Tag weregrown in NZCYM medium (Casein hydrolysate 10.0 g; NaCl 5.0 g; Casaminoacids (Difco) 1.0 g; Yeast extract 5.0 g; MgSO4×7H2O 2.0 g; Maltose 2.0g; Distilled water 1000.0 ml), induced with IPTG(Isopropyl-β-D-thiogalactopyranosid) and after 4 h the cells were lysedby three passages through a french press in a phosphate buffer (100 mM,pH7). The cell lysate was cleared by centrifugation at 10′000 g for 15minutes.

To purify the enzyme, the clear cell lysate was loaded onto a Ni-NTAaffinity column (Qiagen, Hilden, Germany). The column was washed with abuffer containing 20 mM imidazole and finally eluted with a buffer withan increasing concentration of 100-250 mM imidazole. The resultingeluate contained the recombinant enzyme in >95% purity as shown by SDSpage.

Example 4 Heterologous Expression and Production of the K411 Peptidase(SEQ ID NO:4)

The SEQ ID NO: 4 peptidase from Corynebacterium K411 was expressed,produced and purified as described above for SEQ ID: NO:2.

Example 5 Activity of Ax20 and K411 Peptidases

50 ml of recombinant E. coli culture formed as described in example 3and 4 was harvested, resuspended in a final volume of 3 ml of phosphatebuffer and then disrupted with ultrasonication. The soluble extract wascleared by centrifugation. Different dilutions of the resulting extractwere incubated with the cys-gly-conjugate (1 mM) and in presence as wellas in absence of an excess of the β-lyase. As a negative control, clearcell lysate from E. coli transformed with an empty vector, and celllysates with the cys-gly-conjugate but no β-lyase were used.

The table shows the release of the malodourant(3-sulfanyl-3-methyl-hexanol) in mM as determined with the fluorescencetest described in example 1.

As shown in the table below, see columns 2 to 4, the malodourant(3-sulfanyl-3-methyl-hexanol) was only released from thecys-gly-conjugate by the recombinant E. coli strain transformed with theplasmid containing SEQ ID NO:1 or SEQ ID NO:3 (expressing SEQ ID NO:2/Ax20 or SEQ ID NO: 4/K411 peptidase) and only in presence of the3-lyase. K411 peptidase cleaves the cys-gly-conjugate (see column 5)with a lower efficacy than Ax20 peptidase.

release of 3-sulfanyl-3-methyl-hexanol from the Cys-Gly-conjugate [mM]Ax20 Dilution of Negative control Ax20 Ax20 peptidase with K411 K411clear cell (empty vector) + peptidase w/o peptidase + histidine tag +peptidase w/o peptidase + lysate β-lyase β-lyase β-lyase β-lyase β-lyaseβ-lyase  10 fold 0.10 0.10 1.00 0.94 0.10 0.55  100 fold 0.01 <0.01 0.920.88 <0.01 0.19 1000 fold 0.00 <0.01 0.46 0.36 <0.01 0.03

This shows that the isolated nucleotide sequence of SEQ ID NO:1 and SEQID NO:3 codes for a peptidase enzyme with the relevant enzymaticactivity.

As shown, the histidine tag does not have any significant effect onpeptidase activity.

Example 6 Substrate Specificity of Ax20 Peptidase

The recombinant Ax20 peptidase formed as described in example 3(affinity column purified tagged enzyme) was incubated with differentcompounds, in particular potential candidate dipeptide substrates, tocharacterize the substrate specificity and to identify substrates usefulfor screening assays (for example to screen for modulators includinginhibitors).

The table below lists the tested compounds (mostly dipeptides indicatedby their short names, e.g. ala-gly for an alanine-glycine dipeptide) andindicates whether they are cleaved by the peptidase. Z indicates abenzyloxy-carbonyl protecting group at the N-terminus of the peptide.

Cleavage was determined by thin layer chromatography.

Amounts are qualitative with (+++) indicating >80% cleavage of a 1 mMsolution by 0.1 μg/ml peptidase during an incubation for 1 h; (++)indicates complete cleavage of a 1 mM solution by 1 μg/ml peptidaseduring an incubation for 1 h and (+) indicates partial cleavage (20-80%)of a 1 mM solution by 1 μg/ml peptidase during an incubation for 1 h and(−) indicating the absence of any cleavage.

Compounds/Potential substrates Cleavage Cys-gly-conjugate +++H-Cys(Bzl)-Gly-OH (= S-benzyl-Cys-Gly) +++ Ala-Gly ++ Ala-Ala ++Gly-Gly + Pro-Gly + Z-Gly-Gly − Z-Ala − Z-Gly −

Example 7 Method to Identify Peptidase Modulators/Inhibitors

Cleavage of the peptide substrate Pro-Gly by addition of the recombinantAx20 peptidase

(SEQ ID NO: 2) released a unique free NH₂ group which can be derivatizedby fluorescamine (Fluram™, Fluka, Buchs, Switzerland).

Test compounds (for example, potential enzyme inhibitors) are dissolvedin dimethylsulfoxid (DMSO), for example in individual wells of amicrotiter plate.

Ax20 peptidase dissolved in a phosphate buffer is added to theindividual wells of the microtiter plate (1 μg/ml peptidase), andincubated for 5 min to allow equilibration and binding of theinhibitors, if any, to the peptidase.

The substrate Pro-Gly dissolved in phosphate buffer is added to the testcompound/peptidase mix in each well to give a final concentration of 1mM.

Alternatively, another substrate compound may be used. Useful substratesinclude Ala-Ala, Ala-Gly, the Cys-gly-conjugate and S-benzyl-Cys-Gly. Ifthese compounds are used, detection of the cleavage is performed bydetermining the formed free glycine (by Thin-layer chromatography (TLC)or by high performance liquid chromatography (HPLC)), instead of byfluorometry.

After an incubation time of 60 min at 37° C., the free NH₂ group of theglycine released from Pro-Gly is derivatised by adding Fluram dissolvedin acetonitrile to a final Fluram concentration of 1 mM and a finalacetonitrile concentration of 25% v/v, and after 5 min incubation, thefluorescence is determined on a Flex-station (Molecular devices,Sunnyvale, Calif., USA) with an excitation wavelength of 381 nm and anemission wavelength of 470 nm. Controls are run in parallel andsubjected to the same procedure. A positive control contains DMSOwithout inhibitor, a negative control contains no substrate.

% inhibition is calculated based on a comparison with the positivecontrol (0% inhibition/100% activity). Examples of active inhibitorsthus identified are shown in the table below.

Inhibition of release of free NH₂ groups c from Pro-Gly Test compound[microM] [% inhibition] Ethylenediaminetetraacetic acid 62.5 101.4Ethylenediaminetetraacetic acid 31.25 102.6 Ethylenediaminetetraaceticacid 15.625 101.0 o-Phenantrolin 62.5 100.0 o-Phenantrolin 31.25 95.5o-Phenantrolin 15.625 83.9 pyridine-2,6-dicarboxylic acid 62.5 100.9pyridine-2,6-dicarboxylic acid 31.25 92.4 pyridine-2,6-dicarboxylic acid15.625 72.4 ethylendiamine-N,N′-diacetic acid 62.5 93.9ethylendiamine-N,N′-diacetic acid 31.25 81.0ethylendiamine-N,N′-diacetic acid 15.625 65.72,2′-Ethylendithio-diacetic acid 62.5 −5.3 2,2′-Ethylendithio-diaceticacid 31.25 −2.6 2,2′-Ethylendithio-diacetic acid 15.625 0.1

The results in the table show that the compound Pro-Gly is a usefulsubstrate for high-throughput screening. The only NH2 group present isthe one in Gly released through cleavage by the peptidase, allowing fora superior signal to noise ratio.

An inhibitor is usually identified by an inhibition of at least 50%inhibition, for example at a concentration of 1 mM, 100 microM, 10microM, 1 microM, or below.

Example 7b Method to Identify Peptidase Modulators/Inhibitors UsingVarious Peptidase Enzymes

Example 7 may be performed as described with the exception of replacingthe peptidase enzyme and using one or more of SEQ ID NO:4 (K411peptidase), or a homolog to SEQ ID NO: 4 or SEQ ID NO: 2 (Ax20peptidase).

Example 7c Method to Identify Peptidase Modulators/Inhibitors with BroadInhibitory Spectrum

Example 7 may be performed as described with the exception of using twoor more peptidase enzymes selected from SEQ ID NO:2, SEQ ID NO:4, or ahomolog to SEQ ID NO: 4 or SEQ ID NO: 2, subsequently or in parallel.When performed in parallel, the enzymatic reactions may be performed inthe same well using a compatible buffer.

This has the advantage to identify compounds that act on both peptidasesand identify inhibitory compounds with a broad inhibitory activityagainst various bacterial strains.

Example 8 Method to Identify Peptidase Modulators/Inhibitors that areActive Against Peptidase and/or β-Lyase

The example was performed as an alternative to the screening assay asdescribed in example 7, with the following modifications.

As substrates, the cys-gly-conjugate or the compounds of the substratestructure as described herein above are used.

These include (S-benzyl)-cys-gly, (S-benzyl)-cys-ala, or anyS-substituted cys-gly or cys-Ala conjugate, for example, S-ethyl-,S-propyl-, S-butyl-, S-pentyl-, S-hexyl-, S-phenyl-cys-gly or -cys-ala.

Alternatively, substituted ser-ala or ser-gly derivatives are used whichare substituted at the OH group of serine. The same wide variety ofsubstituents at serine are possible as with cysteine.

For the results shown below (S-benzyl)-cys-gly was used.

The peptidase (SEQ ID NO:2) in a concentration of 0.1 μg/ml was used incombination with β-lyase in a concentration of 5 μg/ml; both enzymeswere dissolved in the same phosphate buffer at the same time.

Simultaneously screening inhibitors of both β-lyase and peptidase hasthe advantage of a more effective screen (i.e. both inhibitors of eitherthe peptidase or the β-lyase or both are identified at the same time).Subsequently each screening “hit” (modulation/inhibition of release ofmalodourant) was further analyzed to determine peptidase activity and/orβ-lyase activity separately.

Instead of derivatising the free NH₂ group of the conjugate by addingFluram, the free SH group released from the conjugate by the subsequentaction of both enzymes was derivatised by adding monobromobimane (Fluka,Buchs, Switzerland) dissolved in a NaCO₃ buffer (100 mM, pH 8.8) to givea final concentration of 0.5 mM to 1 mM. After 5 to 15 min incubationthe fluorescence was measured on a Flex-station (Molecular devices,Sunnyvale, Calif., USA) with an excitation wavelength of 385 nm and anemission wavelength of 480 nm.

Alternatively other thiol-derivatizing agents were used includingN-(9-acridinyl)maleimide and fluorescent detection at 359/440 nm.

Active peptidase inhibitors thus identified are shown in the tablebelow.

Inhibition of release of free SH groups from S-benzyl-cys-gly inpresence of c peptidase and β-lyase Test compound [microM] [%inhibition] Ethylenediaminetetraacetic acid 62.5 99.0Ethylenediaminetetraacetic acid 31.25 96.5 Ethylenediaminetetraaceticacid 15.625 99.3 o-Phenantrolin 62.5 93.1 o-Phenantrolin 31.25 79.9o-Phenantrolin 15.625 54.2 pyridine-2,6-dicarboxylic acid 62.5 95.9pyridine-2,6-dicarboxylic acid 31.25 83.4 pyridine-2,6-dicarboxylic acid15.625 49.6 ethylendiamine-N,N′-diacetic acid 62.5 90.4ethylendiamine-N,N′-diacetic acid 31.25 73.1ethylendiamine-N,N′-diacetic acid 15.625 50.22,2′-Ethylendithio-diacetic acid 62.5 −8.7 2,2′-Ethylendithio-diaceticacid 31.25 −9.1 2,2′-Ethylendithio-diacetic acid 15.625 −10.1

Example 9 Method to Identify Broad-Band Modulators/Inhibitors ActiveAgainst One or More Peptidase (SEQ ID NO: 2 and/or NO: 4) and AMRE

In an additional step to example 7 or 8, the identified peptidaseinhibitors were tested for their modulating effect against the AMREenzyme (Na-acyl-glutamine-aminoacylase described by Natsch inEP1258531), thereby identifying dual inhibitors of both metallopeptidaseenzymes involved in malodour release.

Alternatively, the steps can be performed in parallel in presence of theenzymes at the same time.

AMRE is formed as described in example 6 of EP1258531.

The results are listed in the table below.

Inhibition of release of free NH2 groups from N-Lauroyl-glutamine c(catalyzed by AMRE) Test compound [microM] [% inhibition]Ethylenediaminetetraacetic acid 62.5 61.6 Ethylenediaminetetraaceticacid 31.25 59.8 Ethylenediaminetetraacetic acid 15.625 62.4o-Phenantrolin 62.5 96.1 o-Phenantrolin 31.25 81.8 o-Phenantrolin 15.62542.4 pyridine-2,6-dicarboxylic acid 62.5 72.2 pyridine-2,6-dicarboxylicacid 31.25 52.0 pyridine-2,6-dicarboxylic acid 15.625 28.8ethylendiamine-N,N′-diacetic acid 62.5 42.7 ethylendiamine-N,N′-diaceticacid 31.25 7.2 ethylendiamine-N,N′-diacetic acid 15.625 7.42,2′-Ethylendithio-diacetic acid 62.5 19.6 2,2′-Ethylendithio-diaceticacid 31.25 12.7 2,2′-Ethylendithio-diacetic acid 15.625 22.4

Thus the results demonstrate that active inhibitors, in this case metalchelators, active against more than one malodour forming enzyme, can beidentified with a combination of the assays as described, therebyallowing for an effective assay to identify broad-bandmodulators/inhibitors.

Example 10 Differences in Cleavage of the cys-gly-Conjugate when Cleavedwith Ax20 Cell Lysate or β-Lyase

The test compounds (Cys-conjugate (1 mM) or Cys-Gly-conjugate (1 mM))were incubated with β-lyase.

The test compounds (cys-conjugate (1 mM) or cys-gly-conjugate (1 mM))were incubated also with the wild-type strain Corynebacterium sp. Ax20extract naturally containing the peptidase of SEQ ID NO:2.Corynebacterium sp. Ax20 cellular extract was prepared as described inexample 1. The resulting extract was diluted to the same volume as theoriginal culture has had when adjusted to an optical density of OD 4.The substrate was incubated with the extract for 24 h.

Cleavage was measured by gas chromatography to determine the release of3-sulfanyl-3-methyl-hexanol in mM. The results are shown in the tablebelow.

release of 3-sulfanyl-3- release of 3-sulfanyl-3- methyl-hexanol frommethyl-hexanol from cys- cys-conjugate gly-conjugate [mM] [mM] β-lyase0.59 <0.03 C. sp. Ax 20 extract 0.31 0.21

The results show that only the cys conjugate but not thecys-gly-conjugate was cleaved by the recombinant β-Lyase, indicatingthat the β-Lyase cannot cleave the main substrate in the axillasecretion (see row 2 of the table above).

However, the extract from C. sp. Ax 20 isolated from the axilla of ahuman test subject does release 3-sulfanyl-3-methyl-hexanol from bothconjugates (see row 3 of the table above).

Example 11 Separate C. sp. Ax20 Extract Fractions are not able toRelease Malodourant

Cellular extracts obtained from C. sp. Ax20 as described in example 1were fractionated using a Mono-Q anion exchange column. The resultingfractions were separately incubated with the cys-gly-conjugate and therelease of 3-sulfanyl-3-methyl-hexanol (malodourant) was measured asdescribed in example 1.

The results showed that no single fraction of C. sp. Ax20 extract wasable to release 3-sulfanyl-3-methyl-hexanol from the cys-gly-conjugate.This demonstrates that there is no single enzyme present in C. sp.extracts that can release the malodourant from the cys-gly-conjugate.

Example 12 C. SP. Extract Fraction Releases Malodourant when Combinedwith β-lyase

Fractions of C. sp. Ax20 cellular extracts were formed as described inexample 11.

Each fraction was incubated with β-lyase and with the cys-gly-conjugate.The release of 3-sulfanyl-3-methyl-hexanol (malodourant) was measured asdescribed in example 1 above.

The results showed that a single fraction released3-sulfanyl-3-methyl-hexanol from the cys-gly-conjugate in presence ofthe β-lyase. The other fractions or β-lyase on its own were not able torelease the malodourant.

Together with the results of example 11 this demonstrates that twoenzymes are mediating the cleavage of the cys-gly-conjugate: first thepeptidase cleaving the dipeptide between the gly and the cys residue,and then a β-lyase subsequently releasing the malodourant from thecys-conjugate formed by the peptidase.

Example 13 Malodour Release by Corynebacteria Extract is Inhibited byPeptidase Inhibitors

Extracts from C. sp. Ax20 and C. jeikeium K411 were formed as describedin example 1.

O-phenantrolin (0.5 mM), a metallopeptidase inhibitor, or Complete™(EDTA free), a protease inhibitor mix (Roche biochemicals, Switzerland;use concentration as indicated by the manufacturer) that contains broadspecificity serine and aspartate-peptidases inhibitors but nometallopeptidase inhibitors were added to batches of the extracts andincubated for 10 min.

The cys-conjugate or the cys-gly-conjugate (1 mM), both precursors forthe malodourant 3-sulfanyl-3-methyl-hexanol, were then added to the C.sp. Ax20 or C. jeikeium K411 extracts containing the differentinhibitors.

The release of the malodourant 3-sulfanyl-3-methyl-hexanol, whichindicates enzyme activity in the Corynebacteria cell lysates, wasmeasured as described in examples 12 and 13.

The results (in % inhibition of 3-sulfanyl-3-methyl-hexanol releasecompared to reactions without added inhibitors) are indicated in thetable below.

C. sp. Ax20 C. jeikeium K411 Cys-conj. Cys-Gly-conj. Cys-conj.Cys-Gly-conj. o-phenantrolin 0 100 13 93 Complete ™ 1 0 14 37

As shown in example 10, incubation with C. sp. Ax20 extract induced asignificant malodourant release from both conjugates, and C. jeikeiumK411 extract also cleaved both substrates, though at a lower rate.

The addition of the metallopeptidase inhibitor o-phenanthrolin does notinhibit Cys-conjugate cleavage (0% inhibition) but inhibitscys-gly-conjugate cleavage (100% inhibition) when extract from C. sp. isused. Similarly, for C. jeikeium extract, there is no significantinhibition for the cys-conjugate but high inhibition of thecys-gly-conjugate (93%).

This shows that the cleavage of the cys-gly-conjugate (but not thecys-conjugate) is mediated by an enzyme susceptible to inhibition bymetallopeptidase inhibitor o-phenanthrolin.

This also indicates that the β-lyase reaction (Cleavage of theCys-conjugate) is not blocked by o-phenanthrolin, but only the cleavageof the cys-gly bond is inhibited, and shows that a metallopeptidase inextracts from Corynebacteria exclusively catalyses this reaction.

The mixture of protease inhibitors (Complete™) with broad specificityfor different serine and aspartate-peptidases did not significantlyblock cleavage of either conjugate, showing that noserine/aspartate-peptidase is involved in the release of themalodourant.

While the peptidases, nucleotides, expression vectors, host cells,methods and kit have been described above in connection with certainillustrative embodiments, it is to be understood that other similarembodiments may be used or modifications and additions may be made tothe described embodiments for performing the same function(s). Further,all embodiments disclosed are not necessarily in the alternative, asvarious embodiments may be combined to provide the desiredcharacteristics. Variations can be made by one having ordinary skill inthe art without departing from the spirit and scope of the disclosure.Therefore, the nucleotides, polypeptides/peptidases, expression vectors,host cells, methods and kit should not be limited to any singleembodiment, but rather construed in breadth and scope in accordance withthe recitation of the attached claims.

1. A method for identifying a modulator of body malodour formation, themethod comprising the steps of: (i) contacting a peptidase with apeptidase substrate and at least one test agent; and (ii) determiningthe effect of the at least one test agent on the peptidase-mediatedreaction rate, wherein the peptidase has the catalytic activity torelease glycine from the substrate compound of formula FIII

wherein the peptidase is homologous to a polypeptide sequence selectedfrom the group consisting of SEQ ID NO:2 and SEQ ID NO:4 with a sequenceidentity of at least 40%; wherein the peptidase comprises the followingconserved partial sequences: ERDGRWYGRGXADCKG between aminoacid 105 and150, EGSEEXG between aminoacid 150 and 180, HSGXXGGXAPDA betweenaminoacid 205 and 255, GGSIPL between aminoacid 385 and 425; and whereinthe aminoacids are numbered starting from the N-terminus of asubstantially homologous peptidase in its naturally occurring form, andthe letters refer to the single character aminoacid code and X is anyone of the 20 common aminoacids.
 2. The method of claim 1 wherein thepeptidase substrate in step (i) is a compound of formula FI

wherein X is selected from the group consisting of S and O, and whereinR1 is residue selected from H and methyl, and wherein R2 is a residueselected from the group consisting of a straight or branched C1 to C₁₀alkyl, a straight or branched C1 to C₁₀ alkanol, a phenyl, and a benzyl.3. The method of claim 1 wherein the substrate is a compound selectedfrom the group consisting of S-benzyl-Cys-Gly, O-benzyl-Ser-Gly,(1-(2-hydroxyethyl)-1-methylbutyl)-L-cysteinyl-glycine,S-benzyl-Cys-Ala, O-benzyl-Ser-Ala, Pro-Gly, Ala-Gly, Ala-Ala, andPro-Ala.
 4. The method of claim 1 wherein an additional enzyme, acystathionine-β-lyase, is incubated with the substrate and the testagent in parallel or subsequently, and wherein in step (ii), the effectof the test agent on cleavage by the peptidase and β-lyase enzymes isdetermined by the change in formation of at least one of their reactionproducts, optionally a reaction product selected from a thiol reactionproduct and a hydroxy reaction product.
 5. A kit comprising: (i) apeptidase, and (ii) a substrate compound that is cleaved by thepeptidase, for combined use to identify test agents as modulators of thepeptidase and malodour formation, wherein the substrate compoundoptionally is a compound of formula FI

wherein X is selected from the group consisting of S and O, and whereinR1 is residue selected from H and methyl, and wherein R2 is a residueselected from the group consisting of a straight or branched C1 to C₁₀alkyl, a straight or branched C1 to C₁₀ alkanol, a phenyl, and a benzyl,and wherein optionally said substrate compound of formula FI is selectedfrom the group consisting of S-benzyl-Cys-Gly, O-benzyl-Ser-Gly,(1-(2-hydroxyethyl)-1-methylbutyl)-L-cysteinyl-glycine,S-benzyl-Cys-Ala, O-benzyl-Ser-Ala, Pro-Gly, Ala-Gly, Ala-Ala, andPro-Ala, wherein the peptidase has the catalytic activity to releaseglycine from the substrate compound of formula FIII

wherein the peptidase is homologous to a polypeptide sequence selectedfrom the group consisting of SEQ ID NO:2 and SEQ ID NO:4 with a sequenceidentity of at least 40%; wherein the peptidase comprises the followingconserved partial sequences: ERDGRWYGRGXADCKG between aminoacid 105 and150, EGSEEXG between aminoacid 150 and 180, HSGXXGGXAPDA betweenaminoacid 205 and 255, GGSIPL between aminoacid 385 and 425; and whereinthe aminoacids are numbered starting from the N-terminus of asubstantially homologous peptidase in its naturally occurring form, andthe letters refer to the single character aminoacid code and X is anyone of the 20 common aminoacids.
 6. A method of inhibiting a peptidasein its ability to cleave its substrate wherein the peptidase iscontacted with a peptidase inhibitor and wherein the peptidase has thecatalytic activity to release glycine from the substrate compound offormula FIII

wherein the peptidase is homologous to a polypeptide sequence selectedfrom the group consisting of SEQ ID NO:2 and SEQ ID NO:4 with a sequenceidentity of at least 40%; wherein the peptidase comprises the followingconserved partial sequences: ERDGRWYGRGXADCKG between aminoacid 105 and150, EGSEEXG between aminoacid 150 and 180, HSGXXGGXAPDA betweenaminoacid 205 and 255, GGSIPL between aminoacid 385 and 425; and whereinthe aminoacids are numbered starting from the N-terminus of asubstantially homologous peptidase in its naturally occurring form, andthe letters refer to the single character aminoacid code and X is anyone of the 20 common aminoacids.
 7. The method of claim 6 for preventingor reducing the formation of body malodour wherein an inhibitor of apeptidase is applied to a body surface, wherein the peptidase has thecatalytic activity to release glycine from the substrate compound offormula FIII

wherein the peptidase is homologous to a polypeptide sequence selectedfrom the group consisting of SEQ ID NO:2 and SEQ ID NO:4 with a sequenceidentity of at least 40%; wherein the peptidase comprises the followingconserved partial sequences: ERDGRWYGRGXADCKG between aminoacid 105 and150, EGSEEXG between aminoacid 150 and 180, HSGXXGGXAPDA betweenaminoacid 205 and 255, GGSIPL between aminoacid 385 and 425; and whereinthe aminoacids are numbered starting from the N-terminus of asubstantially homologous peptidase in its naturally occurring form, andthe letters refer to the single character aminoacid code and X is anyone of the 20 common aminoacids.
 8. The method according to claim 7wherein the compound is applied in form of a dermatologically acceptablecomposition comprising at least one excipient.
 9. A method for preparinga personal care product having an effect against body malodour formationwherein a peptidase inhibitor is added to a personal care productformulation; wherein the peptidase has the catalytic activity to releaseglycine from the substrate compound of formula FIII

wherein the peptidase is homologous to a polypeptide sequence selectedfrom the group consisting of SEQ ID NO:2 and SEQ ID NO:4 with a sequenceidentity of at least 40%; wherein the peptidase comprises the followingconserved partial sequences: ERDGRWYGRGXADCKG between aminoacid 105 and150, EGSEEXG between aminoacid 150 and 180, HSGXXGGXAPDA betweenaminoacid 205 and 255, GGSIPL between aminoacid 385 and 425; and whereinthe aminoacids are numbered starting from the N-terminus of asubstantially homologous peptidase in its naturally occurring form, andthe letters refer to the single character aminoacid code and X is anyone of the 20 common aminoacids.
 10. A composition comprising apeptidase substrate and an isolated peptidase, wherein the peptidasesubstrate is a compound of formula FI

wherein X is selected from the group consisting of S and O, and whereinR1 is residue selected from H and methyl, and wherein R2 is a residueselected from the group consisting of a straight or branched C1 to C10alkyl, a straight or branched C1 to C10 alkanol, a phenyl, and a benzyl;wherein optionally said substrate compound of formula FI is selectedfrom the group consisting of S-benzyl-Cys-Gly, O-benzyl-Ser-Gly,(1-(2-hydroxyethyl)-1-methylbutyl)-L-cysteinyl-glycine,S-benzyl-Cys-Ala, O-benzyl-Ser-Ala, Pro-Gly, Ala-Gly, Ala-Ala, andPro-Ala; wherein the peptidase is a peptidase with catalytic activity torelease glycine from the substrate compound of formula FIII

wherein the peptidase is homologous to a polypeptide sequence selectedfrom the group consisting of SEQ ID NO:2 and SEQ ID NO:4 with a sequenceidentity of at least 40%; and wherein the peptidase comprises thefollowing conserved partial sequences: ERDGRWYGRGXADCKG betweenaminoacid 105 and 150, EGSEEXG between aminoacid 150 and 180,HSGXXGGXAPDA between aminoacid 205 and 255, GGSIPL between aminoacid 385and 425; wherein the aminoacids are numbered starting from theN-terminus of a substantially homologous peptidase in its naturallyoccurring form, and the letters refer to the single character aminoacidcode and X is any one of the common aminoacids.
 11. The composition ofclaim 10 wherein the peptidase is in a form selected from the groupconsisting of in isolated form, in form of a preparation containingfunctional peptidase, in form of heterologous expression in a suitablehost cell, in form of Corynebacterium jeikeium expressing the peptidase,and in form of Corynebacterium jeikeium K411 expressing the peptidase.12. An isolated peptidase wherein the peptidase is homologous to asequence selected from SEQ ID NO:2 and SEQ ID NO: 4 with a sequenceidentity of at least 40%, wherein the peptidase has the catalyticactivity to release glycine from the substrate compound of formula FIII

wherein the peptidase is homologous to a polypeptide sequence selectedfrom the group consisting of SEQ ID NO:2 and SEQ ID NO:4 with a sequenceidentity of at least 40%; wherein the peptidase comprises the followingconserved partial sequences: ERDGRWYGRGXADCKG between aminoacid 105 and150, EGSEEXG between aminoacid 150 and 180, HSGXXGGXAPDA betweenaminoacid 205 and 255, GGSIPL between aminoacid 385 and 425; and whereinthe aminoacids are numbered starting from the N-terminus of asubstantially homologous peptidase in its naturally occurring form, andthe letters refer to the single character aminoacid code and X is anyone of the 20 common aminoacids.
 13. The isolated peptidase of claim 12wherein the peptidase is homologous to a sequence selected from SEQ IDNO:2 and SEQ ID NO: 4 with a sequence identity of at least 80%.
 14. Anucleotide encoding a peptidase which is selected from the groupconsisting of a nucleotide substantially homologous to a nucleotidesequence of SEQ ID NO:1 as determined by sequence identity; a nucleotidewhich is a conservatively modified variant of SEQ ID NO:1 not causingaminoacid changes when translated into the corresponding protein; anucleotide substantially homologous to SEQ ID NO:1 as determined byhybridisation; wherein the substantially homologous nucleotide asdetermined by sequence identity has a sequence identity of at least 80%;wherein the peptidase has the catalytic activity to release glycine fromthe substrate compound of formula FIII

wherein the peptidase is homologous to a polypeptide sequence selectedfrom the group consisting of SEQ ID NO:2 and SEQ ID NO:4 with a sequenceidentity of at least 40%; wherein the peptidase comprises the followingconserved partial sequences: ERDGRWYGRGXADCKG between aminoacid 105 and150, EGSEEXG between aminoacid 150 and 180, HSGXXGGXAPDA betweenaminoacid 205 and 255, GGSIPL between aminoacid 385 and 425; and whereinthe aminoacids are numbered starting from the N-terminus of asubstantially homologous peptidase in its naturally occurring form, andthe letters refer to the single character aminoacid code and X is anyone of the 20 common aminoacids.
 15. The isolated nucleotide of claim 14wherein said isolated nucleotide forms part of an expression vector. 16.The isolated nucleotide of claim 15 wherein the expression vector formspart of a host cell transfected with the expression vector.
 17. A methodof forming a peptidase comprising the step of: culturing host cellscomprising an expression vector encoding for the peptidase underconditions sufficient for expression, thereby forming the peptidase andoptionally recovering it from the cells, wherein the peptidase has thecatalytic activity to release glycine from the substrate compound offormula FIII

wherein the peptidase is homologous to a polypeptide sequence selectedfrom the group consisting of SEQ ID NO:2 and SEQ ID NO:4 with a sequenceidentity of at least 40%; wherein the peptidase comprises the followingconserved partial sequences: ERDGRWYGRGXADCKG between aminoacid 105 and150, EGSEEXG between aminoacid 150 and 180, HSGXXGGXAPDA betweenaminoacid 205 and 255, GGSIPL between aminoacid 385 and 425; and whereinthe aminoacids are numbered starting from the N-terminus of asubstantially homologous peptidase in its naturally occurring form, andthe letters refer to the single character aminoacid code and X is anyone of the common aminoacids.